Device and methods for the immunological identification of cerebrospinal fluid

ABSTRACT

The present disclosure relates to detection of the presence or absence of cerebrospinal fluid (CSF) in a sample by the detection of one or more antigens that are enriched in CSF compared to their levels in other bodily fluids. The devices and methods are suitable for the detection of the presence or absence of cerebrospinal fluid in samples of mixed bodily fluids from a wide variety of human populations crossing ethnicity, age, gender, health status and genetic variability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/852,761 filed on Aug. 9, 2010, which is a nonprovisional of U.S.Provisional Application No. 61/232,033 filed on Aug. 7, 2009,incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to detection of the presence or absenceof cerebrospinal fluid (CSF) in a sample by the detection of one or moreproteins that are enriched in CSF compared to their levels in otherbodily fluids. Described herein are devices and methods for thedetection of the presence or absence of cerebrospinal fluid in samplesof mixed bodily fluids from a wide variety of human populations crossingethnicity, age, gender, health status and genetic variability.

BACKGROUND

Cerebrospinal fluid (CSF), or liquor cerebrospinalis, is found in thesubarachnoid space as well as in the ventricles surrounding andpenetrating the central nervous system (CNS). CSF bathes the brain andspinal cord and provides hydrative, nutritive, metabolic waste removal,and hydrostatic impact buffer to neurons and glia. CSF is produced fromarterial blood by the choroid plexuses of the lateral and fourthventricles by a combined process of diffusion, pinocytosis and activetransfer. The fluid also contains constituents produced by neurons andglia. After diffusion through the ventricular system into thesubarachnoid space, most of the CSF is reabsorbed by the arachnoidgranulations to reenter the blood stream via the dural venous plexus.Approximately 500 ml of liquor is generated every day; with a totalvolume of 140-150 ml for an adult, the whole CSF is renewed every 6-8hours. The CSF is bounded by the dura throughout the CNS. More fluid isproduced in the rostral CNS and more ultimately drains in the caudalspinal cord to produce a net rostral to caudal fluid flow. CSF is anisotonic mixture mostly of salts, glucose, protein and water. CSF fromthe lumbar region contains 15 to 45 mg/dl protein (0.3-1% of serumprotein concentration) and 50-80 mg/dl glucose (60% of blood glucose).Protein concentration in cisternal and ventricular CSF is lower.

The protein landscape of the CSF can be divided into two groups: Bloodderived proteins, which make up the main fraction in the CSF of healthyindividuals, and brain derived proteins. Approximately 20% of theproteins in the CSF originate from the brain parenchyma, but only asubset of those are actually brain specific.

Despite the fact that the majority of liquor proteins are also found inthe serum, there are multiple sources for proteins unique to the CSF:

Proteins that are released from neurons and glial cells, e.g. tauprotein, S-100, and neuron-specific enolase (NSE).

Proteins released from leptomeniges, e.g. β-trace protein and cystatinC.

Proteins differentially modified by glycosylation or phosphorylationduring synthesis in the choroid plexus, e.g. transthyretin (TTR),angiotensin II, and Insulin-like growth factor II.

There is substantial overlap in the protein profile between CSF andplasma, a considerable number of proteins are unique to the CSF or areuniquely modified by phosphorylation or glycosylation in the CNS.

Lateral Flow Tests, or also known as Lateral Flow ImmunochromatographicAssays or Strip Tests, are designed to rapidly detect the presence orabsence of a given analyte in a heterogenous matrix. A variety ofLateral Flow Tests are currently on the market for home testing, pointof care testing, or laboratory use, for instance pregnancy tests (e.g.,FirstResponse®, ClearBlue®), HIV tests (e.g., OraQuick ADVANCE®,Clearview® Complete), or Chlamydia tests (e.g., Clearview® Chlamydia, inSTlcheck™ Chlamydia).

What is needed is a test suitable for detection of CSF that iscomparable to HIV tests like OraQuick ADVANCE® or Clearview® Complete:It is a point of care test; the test is only qualitative; the operatorneeds minimal training to use the test; the test has an internal controlon the strip to verify accurate sampling.

SUMMARY

In one embodiment, a device for detection of the presence or absence ofcerebrospinal fluid in a sample comprises

a sample application region,

a sample labeling region comprising a first antibody to a CSF-enrichedprotein,

wherein the first antibody is conjugated to a mobile particle;

a sample detection region comprising a second antibody to theCSF-enriched protein, wherein the second antibody is fixed to the sampledetection region,

wherein the presence of a detectable band in the second region indicatesthe presence of cerebrospinal fluid in the sample.

In another embodiment, a method for detecting the presence or absence ofCSF in a sample, comprises

contacting the sample with a binding partner specific for a CSF-enrichedprotein, and

detecting binding partner-CSF enriched protein complexes if present,wherein the presence of detectable complexes indicates the presence ofCSF in the sample.

In the foregoing embodiments, the CSF antigen is Isoform 1 of Neuralcell adhesion molecule-like (SEQ ID NO: 1; Accession Number gi:62088238)protein; Chain A, Human Mesotrypsin Complexed With Bovine PancreaticTrypsin Inhibitor (Bpti) (SEQ ID NO:2; Accession number gi:162330095);CNTN2 Contactin-2 precursor (SEQ ID NO: 3; Accession Number gi|4827022);CNTN1 Isoform 2 of Contactin-1 (SEQ ID NO: 4; Accession Numbergi:28373119); cDNA highly similar to SPARC-like protein 1 (unnamedprotein product) (SEQ ID NO: 5; Accession Number: gi|194388050); NRCAMprotein (Neuronal cell adhesion molecule)[Homo sapiens] possiblyslightly longer fragment (˜96 kDa) (Accession Number: SEQ ID NO: 6;gi|68534652 and SEQ ID NO: 7; gi|109731501); NCAM2 Neural cell adhesionmolecule 2, isoform CRA_a (SEQ ID NO: 8; Accession Number gi|119630409);SERPINA3 serpin peptidase inhibitor, Glade A, member 3 precursor/Isoform1 of Alpha-1-antichymotrypsin/growth-inhibiting protein 25 [Homosapiens] or slightly longer fragment of alpha-1-antichymotrypsinprecursor (SEQ ID NO: 9; Accession Number gi|46981961); AGTAngiotensinogen (SEQ ID NO: 10; Accession Number gi|553181);Angiotensinogen precursor (Serpin A8) (SEQ ID NO: 11; Accession Numbergi|4557287); unnamed protein product also called immunoglobulinsuperfamily, member 4B; in humans, also called cell adhesion molecule 3(SEQ ID NO: 12; Accession Number gi|187608363); cDNA FLJ59893, dickkopfhomolog 3 precursor (SEQ ID NO: 13; Accession Number gi|40548389);SERPINF1 serine (or cysteine) proteinase inhibitor, Glade F (alpha-2antiplasmin, pigment epithelium derived factor, Pedf), member 1 isoform4 factor (SEQ ID NO: 14; Accession Number gi|15988024); human proteinsimilar to GC Vitamin D-binding protein PREDICTED: vitamin D-bindingprotein [Pan troglodytes] (SEQ ID NO: 15; Accession Number 181482); CD14Human monocyte antigen CD14 (CD14) (SEQ ID NO: 16; Accession Numbergi|117646212); CADM3 Homo sapiens cell adhesion molecule 3 (CADM3),transcript variant 1 (SEQ ID NO: 17; Accession Number gi|90080503; SEQID NO: 18; gi|187608363 (human); Neural cell adhesion molecule variant(SEQ ID NO: 19; Accession Number gi:62088238); unnamed protein similarto CLU cDNA FLJ57622, highly similar to Clusterin (SEQ ID NO: 20;Accession number gi|189054091); protein highly similar to Clusterin (SEQID NO: 21; Accession number gi|193787502); LMAN2 Vesicularintegral-membrane protein VIP36 (SEQ ID NO: 22; Accession numbergi|157834800); clusterin isoform 1 [Homo sapiens] (SEQ ID NO: 23;Accession number NM_(—)001831.2); superoxide dismutase 3, extracellularprecursor (SEQ ID NO: 24; Accession number gi|118582275); fibrin alpha Cterm fragment (SEQ ID NO: 25; Accession number gi|223057); Chain A,Human Kallikrein 6 (Hk6) Active Form or KLK6 Isoform 1 of Kallikrein-6(SEQ ID NO: 26; Accession number gi|21465970); APCS Serum amyloidP-component/Chain A or Pentameric Human Serum Amyloid P Component (SEQID NO: 27; Accession number gi|576259); FAM3C Protein FAM3C/family withsequence similarity 3, member C precursor [Homo sapiens] note=“predictedosteoblast protein; interleukin-like EMT inducer (SEQ ID NO: 28;Accession number gi|55629272); protein similar to unnamed proteinproduct [Macaca fascicularis] also called immunoglobulin superfamily,member 4B; in humans, also called cell adhesion molecule 3 (SEQ ID NO:29; Accession number gi|187608363); a CSF-enriched phosphorylated ordephosphorylated form of the foregoing CSF antigens; or a combination oftwo or more of the foregoing CSF antigens.

In another embodiment, a method for the detection of a reactant in abody fluid, tissue or microorganism comprises contacting the body fluid,tissue or microorganism with two or more antibodies, wherein eachantibody specifically reacts with an antigen in the reactant, whereinreaction with each individual antibody does not indicate a positive testfor the reactant, and wherein reaction with the two or more antibodiesindicates a positive test for the reactant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Lateral Flow assay. Analyte is added to the left end of the stripeither by a dropper or by direct dipping. The liquid (around 75 μl) iswicked across the strip to the right. The conjugate pad contains solubleIgG attached to a visible particle (i.e., gold or latex microspheres).If the analyte solution contains the analyte, the antibodies conjugateand the complex migrates across the strip. The mixture first encountersthe test strip, which contains immobilized antibody against the analyte.The analyte, soluble primary and visible tag, then bind to the testline. If no analyte is present the soluble faction passes over the testline. Whether the analyte is present or not, excess soluble IgG bound toindicator binds to the immobilized anti-globin IgG bound to the controlstrip.

FIG. 2 shows advantages of a multi antigen approach to CSF detection.The upper figure represents single antigen assay results for varioustest conditions and the bottom figure shows results of the multi antigenassay. The bars along the X axis represent different assay conditionsand the Y axis represents the degree of immunoreactivity seen by theassay. The upper shaded zone indicates a positive colorimetric responseon the test line of the lateral flow assay. Assays with immunoreactivitythat enters the shaded zone will produce a positive test result. Bar 1:CSF Bars in the upper graph illustrate immunoreactivity of the singleantigen being sufficient to produce a positive test result.Alternatively in the multiple antigen graph (lower) a combination ofantigens, each producing a partial signal accumulates to producepositive assay result. Bar 2: CSF contaminated with blood produces asimilar positive response with a smaller but additive bloodimmunoreactivity (upper bar with thick border). Bar 3: Unusual CSF/bloodsample in which antigen 1 is poorly immunoreactive. In the singleantigen assay, the assay produces a false negative, while the multiantigen assay is still above assay threshold as a result of the otherfive antigen immunoreactivities being intact. Bar 4: CSF/blood with noantigen 1 immunoreactivity. Same results as in Bar 3. Bar 5: No CSF butblood borne cross-reactive antigen. In this case the single antigenassay produces a false positive, but as the immunoreactivity of thesingle antigen is not sufficient to produce a positive signal in themulti antigen assay the assay reports the correct negative result. Bar6: No CSF but blood level of antigen 1 pathologically high. Singleantigen assay produces false positive reacting to heightened bloodlevels. Multi antigen assay reacts to pathogentic antigen 1 levels inblood but does not reach threshold for false positive. This assay isshown with 5 antigen/antibody1/antibody2 mixes, however otherembodiments could contain between 2 and as many as 10antigen/antibody1/antibody2 mixes.

FIG. 3: Two dimensional gel electrophoresis of CSF and blood proteins.An example of a single experiment in which 100 μg of Cy-tagged CSFprotein (A) and 100 μg of Cy3-tagged blood proteins (B) are separated intwo dimensions. A and B are grayscale images of the same gel usingdifferent excitation and emission settings. The pH range is 4-8. C) isthe RGB merge of the two channels with yellow spots indicatingsignificant overlap. D) is an automated extraction of spots with >5×enrichment in either the CSF or blood. All samples were 2× depleted ofmajor serum/CSF proteins (see Methods).

FIG. 4: Liquid chromatography-mass spectroscopy analysis of some of theCSF-enriched spots seen on the gel in FIG. 3.

FIG. 5: CSF-enriched proteins FUSS and dickkopf homolog 3 precursor(DKK3). A) Immunoblot of FLJ55. Affinity purified polyclonal rabbitanti-human antibody produced against a recombinant fragment of FUSSproduces immunoreactivity at the correct molecular weight in the CSFsample but not in the serum sample. B) Affinity purified polyclonalrabbit anti-human antibody produced against a recombinant fragment ofDKK3 also produces immunoreactivity at the correct molecular weight inthe CSF sample but not in the serum sample. In both cases excessiveserum protein was loaded at levels higher then that of the sera. C) Fourseparate samples of CSF indicating immunoreactivity for DKK3 with adifferent affinity purified antibody (left). Five blood samples fail toproduce immunoreactivity. Lane 5 blood is high non specific background.

FIG. 6: Phosphorylated forms of angiotensinogen that are highly enrichedin the CSF. An RGB merge of the Cy3 blood (green) and Cy5 CSF (red). Wehave identified several novel and non-overlapping phosphorylatedversions (right four red spots) that are not present in the blood. Atleast three other combinations (left three spots) are present in bothCSF and blood.

FIG. 7: CSF specific post translational modifications. Change in the CSF2D gel protein distribution pattern before (top panel) and after (middlepanel) removal of all secondary modifications of the extracted proteins.Red spots in lower panel indicate a reduction in a particular proteinsignal following removal of the post-translational modification.

FIG. 8: Experimental flow chart for the production of CSF detection teststrips.

FIG. 9: CSF proteins that are phosphorylated. A single DIGE gel in whichtwo samples of serum protein depleted CSF was run. A) the Cy3 labeledproteins from the CSF sample which was incubated in alkaline phosphatasefor one hour. B) Equivalent sample of serum protein depleted CSF nottreated with alkaline phosphatase. C) Computer generated difference(blue boundaries) between spot volume of the two gels (A vs B). All bluespots represent phosphorylated CSF proteins.

DETAILED DESCRIPTION

Described herein are proteins that are enriched in CSF compared to otherbodily fluids and methods for the detection of the presence or absenceof cerebrospinal fluid (CSF) in a sample by the detection of theseproteins. Also described herein are devices and methods for thedetection of the presence or absence of CSF in samples of mixed bodilyfluids from a wide variety of human populations crossing ethnicity, age,gender, health status and genetic variability. The CSF-enriched proteinsare detected with a specific protein binding partner such as anantibody, a ligand, a receptor, and the like. Binding partners can benatural or synthetic binding partners.

Binding can be detected either directly, or indirectly, such as with afluorescent label attached to the binding partner. While severalembodiments are included that use antibodies as binding partners, itshould be understood that other binding partners can be used in place ofantibodies.

In certain embodiments, the level of the CSF-enriched protein isquantitated. Such quantitation is particularly useful in theidentification of brain injury. Quantitation can be performed by using abinding partner with a detectable label. “Detectable moiety” or a“label” refers to a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Usefullabels include ³²P, ³⁵S, fluorescent dyes, electron-dense reagents,enzymes (e.g., as commonly used in an ELISA), biotinstreptavadin,dioxigenin, haptens and proteins for which antisera or monoclonalantibodies are available. The detectable moiety often generates ameasurable signal, such as a radioactive, chromogenic, or fluorescentsignal that can be used to quantitate the amount of bound detectablemoiety in a sample. The detectable moiety can be incorporated in orattached to a binding partner either covalently, or through ionic, vander Waals or hydrogen bonds. The detectable moiety may be directly orindirectly detectable. Indirect detection can involve the binding of asecond directly or indirectly detectable moiety to the detectablemoiety.

In some embodiments, CSF detection is performed using a lateral flowassay, employing for example, antibodies specific for the CSF protein ofinterest. A lateral flow assay can be a single antigen assay or amultiple antigen assay. In one embodiment, a multiple antigen test usesall of the antigens together to provide a single easy to read answer(i.e., a single band on a strip assay). In another embodiment, amultiple antigen test qualifies or quantifies each of several antigensindividually to give a more complex profile of the antigens that arepresent. Such a profile may be useful to determine the severity of ahead injury, that is, the head injury is less severe when certainCSF-specific proteins are present and more severe when otherCSF-specific proteins are present or levels of each protein provides adegree of injury

Single Antigen Assay:

While lateral flow technology has been successfully used in manyclinical assays, the unique and innovative approach described hereinextends the technology to i.) bind single or multiple CSF-enrichedproteins, thereby increasing sensitivity and specificity of the test,and/or ii.) detect a CSF-specific post-translational modification (e.g.,phosphorylation).

As used herein, a CSF-enriched protein or CSF antigen or polypeptide isan antigen or polypeptide that is specific for CSF or substantiallyenriched in CSF compared to other bodily fluids. Table 1 identifiesseveral proteins known to be concentrated in the CSF. These are notproteins identified in the current application, although they can, insome embodiments, be combined in an assay with one or more proteinsidentified herein in a multi-antigen assay.

TABLE 1 Protein MW (kDa) CSF concentration CSF/serum ratio β-traceprotein  25 16.6 mg/l 34:1 Cystatin C 13.3 3.1 mg/l  5:1 Tau-protein55-74 0.2 μg/l 10:1 S-100 B  21 1.5 μg/l 18:1 NSE  78 8 mg/l  1:1Transthyretin  55 17 mg/l  1:18 Albumin  67 245 mg/l   1:205 IgG 150 25mg/l   1:440

Described herein are proteins that are present in sufficient quantitiesand enriched significantly in CSF compared to their levels in otherbodily fluids, to act as a marker of CSF. The proteins found in pooledsamples of CSF were compared to the proteins in blood, nasal fluid,saliva, sweat, tears and ear effluents (referred to as ‘other bodilyfluids’). CSF from a range of ages (1-70 years) and from both males andfemales was examined. Prior to comparative 2D gel electrophoresis, allfluids were treated to remove dominant serum proteins that are presentin most bodily fluids (i.e., albumin, IgG, etc.). The remaining proteinsfrom CSF and another bodily fluid were differentially tagged with Cy3and Cy5 and run on two-dimensional PAGE. Using this approach, a novelset of proteins which are highly concentrated in the CSF over otherbodily fluids were identified. CSF-enriched secondary modified proteins(i.e., phosphorylated) have also been identifled. Dephosphorylation ofCSF extracts confirmed that the CSF unique spots represent differentialmigration in the isoelectric dimension based on phosphorylation.

In one embodiment, the proteins that are enriched in CSF are used todetect CSF in an assay, such as a lateral flow assay. A lateral flowsystem consists of overlapping membranes containing the dried componentsneeded for the test performance (FIG. 1). These membranes are assembledto small strips which can be placed into a plastic housing for betterhandling. The patient's material is loaded to the Sample Pad. In thecase of whole blood/capillary blood samples a separation of blood cellsand plasma takes place. The liquid fraction of the patient's samplediffuses through the Conjugate Pad containing labeled antibodies, whichare specifically directed against the analyte of interest. Theantibodies (conjugate) are re-dissolved and the analyte is specificallybound by the gold (or latex) conjugate. The analyte-gold-conjugatecomplex further diffuses through the Analytical Membrane. On thismembrane two lines are arranged one after the other: (i) the Test Linecontaining a second set analyte-specific antibodies responsible forimmobilizing the analyte-gold conjugate complexes and (ii) the ControlLine fixing non-bound gold antibodies indicating that the conjugate hasoverflown the Test Line. If the analyte of interest is available abovethe detection limit the Test Line and the Control Line are clearlyvisible; if the analyte is below the detection limit only the ControlLine appears during test time. The last component of the rapid test isthe Wicking (or Sink) Pad which simply collects the fluid miming throughthe test system and preventing backflow of the fluid through the testsystem.

Lateral Flow Immunochromatographic Assays are designed either assandwich assays or as competitive assays. Sandwich assays makes use oftwo different antibodies raised against the same analyte, one to colorthe analyte and one to concentrate the analyte at the test line. Thetest line will show as a colored band in positive samples. Competitiveassays provide already colored analyte on the test strip and a set ofantibodies against the analyte at the test line. The sample flows withthe provided colored analyte towards the test line and competes forantibody binding. The test line will show as a colored band in negativesamples.

CSF Assay Design Specifications:

The assay described herein can be used to accurately identify traces ofCSF when it is mixed with a variety of non-CSF bodily fluids. These‘other fluids’ are, for example, nasal and ear effluents, saliva, tears,sweat, urine and blood. The assay is intended to minimize false positiveor false negative results regardless of the physiologic, metabolic orpathologic state, gender, age or ethnicity of the subject.

In one embodiment, the limit of detection is >5% CSF in a pure fluid ormixture of any of the above fluids. It may be possible to achieve ahigher sensitivity but it will be essential to maintain the specificityin addition to the increased sensitivity. Thus, in some embodiments, alimit of detection of >1% CSF is achieved.

Multi Antigen CSF ‘Tissue’ Assay:

In one embodiment, the assay is one that will allow the detection of thepresence of CSF via simultaneous detection of multiple CSF-enrichedproteins. That is, the test includes two or more markers for CSF toprovide improved reliability of CSF detection. Rather than testing for asingle tiomarker', the multiple marker assay will be robust and providethe correct answer under a variety of potential and unknowncircumstances with high selectivity and sensitivity. For example, asingle antigen assay may produce a false positive if the antibodyrecognizes an antigen in a fluid other than CSF (i.e. blood). If theassay tests for a antigen which is ‘enriched’ in CSF but not ‘exclusive’to CSF, an aberrantly high blood level could produce a false positive.This may be problematic because it is not feasible to test the stripunder all possible physiologic, pathologic, ethnic, sex, dietary,age-related, etc. conditions to look for false results. Further, thelevel of particular CSF antigen may be reduced below detection level, ora particular CSF antigen may have a rare genotypic difference, thusreducing reactivity in certain human populations thereby producing afalse negative. These are all potential difficulties that arise frombasing a test on a single CSF-enriched antigen (see FIG. 2). The novel‘Multi antigen’ assay for detecting CSF in mixed body fluid samplesshould provide substantial improvement over single-antigen tests. Incertain embodiments, the multi-antigen test includes at least oneantibody specific for each of 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigens thatare enriched in CSF compared to their levels in other bodily fluids. Inother embodiments, at least two antibodies specific for each antigen areemployed.

As described herein, a large number of CSF-enriched protein spots havebeen extracted and analyzed by LC-MS. The rationale for this approach isillustrated in FIG. 2. Several CSF-enriched antigens have beenidentified and at least two different antibodies have been produced toeach antigen. Mixtures of each of the two sets of IgG are added to themobile and immobilized portions of the test strip (see FIG. 2),respectively. The multi antigen assay works by applying a concentrationof antibodies for a particular antigen that are below the threshold fordetection when all antibody molecules are bound. A mixture of severalantibodies each a subthreshold levels are utilized in the assay. WhenCSF is added, all antibodies bind and accumulate producing a positivesignal. The optimal embodiment would use at least 5-6 different antigenswith a detection threshold of 4 so loss of a single antigen will notcause a false negative. In one embodiment, the device or test comprises4 to 10 different antibodies that each specifically binds a differentCSF antigens, wherein a positive test does not require binding to allantibodies. Accumulation of IgG/antigen on the test strip is linear andsubthreshold levels for individual detection of each antibody are usedthen only the addition of other positive antibodies will produce apositive reaction. A positive response requiring accumulation of atleast 4 IgG/antigens the assay will be more robust in the face offluctuations in the levels of any one antigen. The assay will also bemore robust in the face of aberrant increases in single antigenimmunoreactivity in contaminating bodily fluids. Artifactualimmunoreactivity of 1-3 of the antigens will not produce a positivetest, therefore the test will be more robust and produce fewer falsepositives.

Identification Of CSF-Enriched Proteins:

CSF samples from 1-40 individuals are pooled and 200 μl of the pooledsamples are analyzed. Samples of sera from 1-40 individuals are pooledand 1 ml of pooled sera are analyzed. Major proteins shared by the bloodand CSF (i.e. albumin, immunoglobins, etc.) were removed from bothsamples by repeated affinity chromatography.

In vitro label 50 μg of the control protein extract and 50 μg of theexperimental protein extract with GE Healthcare Cy-3 and Cy-5N-hydroxysuccinimidyl ester dyes. These dyes have been matched withrespect to charge and mass—with the single positive charge of the dyereplacing the charge lost by the modified lysine or N-terminus of theprotein. Cy-3 and Cy-5 labeled proteins co-migrate—with the dye labeladding approximately 450 Da to the proteins in each sample.

Control, experimental, and internal standard samples were mixed together(i.e., 150 μg total protein) and then an equal volume of 2× SampleBuffer added.

The volume was brought up to 450 ul with Rehydration Buffer Immobiline™(IPG) Drystrips (GE Healthcare) 24 cm were rehydrated for 10-24 hrs, andisoelectric focusing carried out. We used a number of different pHranges including: 3-7, 4-7, 3.5-4.5, 4.0-5.0, 4.5-5.5, 5.0-6.0, 5.5-6.7,and 6-9. SDS polyacrylamide gel electrophoresis (second) dimension wascarried out on a 10 inch wide by 7.5 inch tall by 1.0 mm thick gel withone side coated with Gelbond®. We used a 12.5% polyacrylamide gel whichwill optimally separate 12-100 kD proteins.

Immediately after SDS PAGE, the gel (which is still held between twoglass plates) was scanned at all 3 wavelengths simultaneously on the GEHealthcare Typhoon™ 9410 Imager. After scanning, 16 bit TIFF files ofeach color channel were exported for image analysis using thedifferential in-gel analysis module of the GE Healthcare DeCydersoftware package. After spot detection (which includes automaticbackground correction, spot volume normalization and volume ratiocalculation), a user defined “dust filter” was applied to each gel. Thishas the effect of automatically removing non-protein spot features fromthe gel and is followed by recalculation of experimental parameters.

The front glass plate was removed and the gel was then fixed and stainedwith Sypro Ruby, which is the fluorescent stain that was used as a guideto excise spots of interest from the gel. The reason for using SpyroRuby, which stains all protein in the gel, is that the Cy-dye labelingis carried out such that the extent of incorporation will be <5% interms of mole Cy-dye/mole protein. Since the Cy-dye has a MW of about580 Da, low MW proteins (e.g., 10 Kd) labeled with Cy-dyes will notexactly co-migrate in the SDS PAGE dimension with their non-labeledcounterparts.

GE Healthcare DeCyder™ software was used to quantify the gel image andto identify a “pick list” of differentially expressed protein spots tobe excised and subjected to MS-based protein identification. TheDeCyder™ software can analyze any two Cy-dyed gel images, either on thesame gel or on different gels, match the spots between the two images,and then identify differentially expressed protein spots. The DeCyder™software automatically outputs a listing of statistically significantdifferences in protein expression including t-test values, using theCy-2 internal standard. Differentially expressed spots were identifiedusing a number of criteria including area, volume, 3D peak slope, 3Dpeak height, and/or statistical variation. Protein spots that showdifferent degrees of intensity between the two samples were highlightedby the software and confirmed manually. The DeCyder™ software was alsoused to analyze Sypro Ruby images, match the spots found with Syprostaining to those identified with the Cy-dye stains, and then choose a‘pick list’ from the Sypro stained gel image.

The protein spot pick list was transferred to the Ettan™ Spot Pickerinstrument (GE Healthcare) which automatically excised the selectedprotein spots from the gel and transferred them into a 96-wellmicrotiter plate.

The excised protein spots were then subjected to automated in-geltryptic digestion on the Ettan™ TA Digester.

An aliquot of each digest was spotted (along with matrix) onto aMALDI-MS target.

High mass accuracy, automated MALDI-MS/MS spectra were acquired on eachtarget (using an Applied Biosystems 4800 T of/T of instrument) and theresulting peptide masses were subjected to database searching usingMascot algorithms.

The remaining aliquots of digests of protein spots that are notidentified by this approach were subjected to nanospray or LC/MS/MSanalysis (Micromass Q-T of) with the resulting MS/MS spectra then beingsubjected to Sequest database searches to identify proteins present inthe sample.

CSF-Enriched Protein Phosphorylation Sites as Antigens for a CSF TestStrip:

During the course of Fluorescence Difference Gel Electrophoresis (DIGE)experiments to identify CSF-enriched proteins, spots distributed in thepH dimension that were highly CSF-enriched (i.e. not present in bloodsamples) were identified, however upon protein identification by LC-MS,it was established that many of these proteins were in fact present inthe blood but had a different patterns in the pH dimension of the gel(FIG. 6). Regularly spaced spots of the same molecular weight oftenrepresent differentially phosphorylated versions of the same protein.The differential and regular migration in the pH dimension is indicativeof the large but quantal nature of the negative charge on the PO₃ ⁻groups. Upon phosphopeptide mapping of these spot arrays, it wasdetermined that this was in fact the case. Several of these proteins(including angiotensinogen, (FIG. 6) had highly CSF-enrichedphosphorylations. In some cases these phosphorylation sites wereserine/threonine phosphorylations, and in other cases they were tyrosinephosphorylations. In all, proteins were selected with multipleCSF-enriched sites per protein (i.e. angiotensinogen). As it is possibleto produce antibodies that recognize a single epitope only whenphosphorylated, phosphorylation sites will be included as antigens inthe assays described herein. These phosphorylated epitopes areattractive as candidates as they are very prevalent and the presence oftwo CSF-enriched phosphorylation sites on a single protein opens thedoor to making pairs of antibodies to different sites that can be useddifferentially on the mobile and immobile regions of the strip torequire dual phosphorylation for a positive response. We have run DIGEgels comparing CSF proteins that have been dephosphorylated withalkaline phosphatase (FIG. 9). This has identified proteins listedherein as differentially phosphorylated in the CSF.

Identification of antigens is performed using 2 dimensional DIGE gelelectrophoresis followed by trypsin digestion and LC-MS. The dominantproteins in both blood and CSF are removed by affinity columns prior toelectrophoresis. These proteins are ubiquitously present in bodilyfluids (i.e. albumin, immunoglobins etc.). We run all samples doublyacross columns to remove 14 dominate serum proteins. We run theextracted proteins from 1-2 mls of whole blood on gels along withproteins from 200 μl of CSF. This enriched the blood proteins to ensurewe are identifying proteins that are enriched in the CSF. Proteins fromthe CSF are labeled using either Cy3 or Cy5 fluorophores. In contrastblood proteins are labeled with either Cy5 or Cy3, respectively. Thesamples are then mixed and loaded on a 2 dimensional PAGE gel. Numerousdifferent gels are run focusing on different regions of the molecularmass dimension (Y-dimension) and pH dimension (X-dimension). Followingrunning of the gel, the intensity of the differentially visualizedfluorescently labeled proteins are quantified and compared by anautomated computer program. Those spots that are enriched by at least 5×in the CSF are robotically collected, trypsin digested and analyzed byLC-MS. Peptide molecular weights are compared to published databases.Enriched proteins are selected as candidates and standard molecularbiologic methodology are employed for the production of Histidine-taggedrecombinant proteins in bacteria or alternatively peptides correspondingto specific regions of the proteins are produced synthetically.Monoclonal and polyclonal antibodies are produced by a commercial houseusing provided antigens. Affinity purification is performed by standardcolumn techniques utilizing cyanogen bromide-activated columns andrecombinant proteins used for immunization. CSF-specific antigens areidentified by trypsin and chymotrypsin digestion followed by LC-MS andphosphopeptide determination.

Validation of CSF-enriched antibodies is conducted by separatingdiscrete volumes of whole bodily fluid proteins on SDS-PAGE,transferring to nitrocellulose membranes, immunoblotting first withprimary antibodies against the antigens and then HRP-labeled secondaryantibodies followed by ECL quantification. Antigens that have a >5×immunoreactivity in CSF over levels larger volumes of whole blood, nasaland ear effluents, tear, saliva or sweat are pursued. Samples of bodilyfluids from 20 to 30 different individuals of each are tested for eachantigen. Fluid samples are purchased from commercial laboratories thatassure purity or directly collected. Bodily fluids are tested fromindividuals ranging in age from infants to elderly (75 years), male andfemale, as well as several common pathological conditions (i.e. advancedstage diabetes, coronary artery disease, asthma, etc.).

To identify phosphorylation state specific antigens, two-dimensionalgels are produced as described above however three labeled proteinfractions are produced (Cy2, Cy3 and Cy5): CSF, whole blood and CSFproteins in which all protein phosphorylations have been removed byalkaline phosphatase in an additional step prior to labeling. Acomparison is then made between the dephosphorylated and normal CSFchannels for alterations. Spots that disappear followingdephosphorylation and are not present in the blood protein fluorescencechannel are collected and sequenced. Absolute identification of the siteof phosphorylation is determined by phospho peptide and phospho aminoacid analysis, in vitro phosphorylation of recombinant proteins andprotein fragments and immunoreactivity with phosphostate specificantibodies.

Once antibodies have been selected for use in the test strips, therelative affinity of each of the antibodies will be determined byrunning dilution curves using pure samples of recombinant antigens. Thiswill guide the mixing of antibodies for inclusion on test strips.

In one embodiment, included herein are devices and methods for rapid,bedside or triage site testing of bodily fluids, surgical sites orwounds for the presence of cerebrospinal fluid. In another embodiment atest is proposed that allows detection of CSF enriched proteins insamples of blood, plasma or sera as an indication of central nervoussystem (CNS) injury, breach or damage. Tests can include a single ormultiples of the antigens described herein as markers of damage to theCNS. Described herein are newly-identified CSF-specific or enrichedantigens that can be used individually or in combination to detect CSFin a broad spectrum of individuals from pediatric to geriatric, anddespite the presence of diseases, personal habits, or individual geneticvariability that might alter the composition of bodily fluids.

In one embodiment, included herein are devices for the detection ofcerebrospinal fluid in samples such as those suspected of containingcerebrospinal fluid, wherein the devices include one or more antibodiesspecific for one or more CSF antigens as described above. The CSFantigens can be employed in combinations to enhance the signal to noiseratio and to overcome individual variability in the expression of theanti-gens described above in different bodily fluids. In someembodiments, the detection of multiple antigens provides superiorsensitivity and selectivity over detection of a single CSF-enrichedantigen.

In one embodiment, described herein are devices for the detection ofcerebrospinal fluid in samples such as those suspected of containingcerebrospinal fluid, wherein the devices include one or more antibodiesspecific for one or more CSF antigens in a state of post-translationalmodification that is specific to the cerebrospinal fluid anddistinguishable from the same antigen in other bodily fluids by virtueof the post-translational modification.

In some embodiments, described herein are devices for the detection ofcerebrospinal fluid in samples such as those suspected of containingcerebrospinal fluid, wherein the devices include one or more antibodiesspecific for one or more CSF antigens in a state of phosphorylation thatis specific to the cerebrospinal fluid and distinguishable from the sameantigen in other bodily fluids by virtue of the phosphorylation.

Samples for testing using the devices disclosed herein can be taken fromdifferent sites in the human body, such as at a site of surgery (i.e.head, neck, ear, throat, nasal or spinal surgeries) where the potentialfor CSF leakage is possible; at a site of epidural injection or spinaltap; or at a site of wounds in areas where a breach of the meninges ispossible (i.e. head, neck, spinal cord, nasal compartment, nose, ears,throat, skull, etc.), or where the injured party demonstrates signs ofpossible meningeal breach or serious injury to the central nervoussystem; or at a site of epidural injection, spinal injection or spinaltap. The antigens identified herein are particularly good markers forbrain injury. Additional samples include saliva and urine samples.

The unique approach of performing 2D-DIGE studies to compare thecomponents of human CSF and serum has yielded a number of antigens thatare specific to, or highly enriched in CSF. Antibodies specific forthese antigens are markers of the presence of CSF in bodily fluids, orat wound, surgical or injections sites where its presence would beatypical and potentially threaten the health or life of a patient ortrauma victim.

In some embodiments, the above-described CSF antigens havepost-translational modifications such as phoshorylation, glycosylation,sumoylation, ubiquitination, lipidation, nitrosylation, acetylation,neddylation, where those post-translational modification are specific tothe CSF form of the antigen may be used by the lateral flow assay,western blots, ELISA or immunoprecipitation.

In some embodiments, multiple antigens may be used and may includecombinations of antibodies that detect simple antigens (i.e., unmodifiedantigens) with antibodies that detect post-translationally modifiedantigens such as described above and in any of the various assays,lateral flow, Western blot, ELISA, or immunoprecipitation.

In one embodiment, antibodies are used to determine if a sample containspolypeptides associated with the presence of CSF indicating the presenceor absence of CSF. Antibody binding is detected by, for example,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, surface plasmon resonance,immunocytochemistry, immunohistochemistry, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, and the like. Detection of antibodybinding can be achieved using enzymatic, colorimetric, fluorescent,bioluminescent, luminescent, colored latex beads, colloidal gold and/orsilver methods.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many methods are known in the art for detecting binding in animmunoassay.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a score correlating to the presenceof specific polypeptides and likelihood of CSF in a sample based on theresult of the immunoassay is utilized.

In other embodiments, the immunoassay is as described in U.S. Pat. Nos.5,599,677 and 5,672,480, each of which is herein incorporated byreference.

Provided herein are isolated antibodies or antibody fragments (e.g., Fabfragments, Fab₂ fragments, and the like). Antibodies can be generated toallow for the detection of polypeptides associated with the presence ofCSF. The antibodies are prepared using various polypeptides, syntheticpeptides and/or recombinant proteins associated with the presence of CSFand fragments thereof. In one embodiment, the immunogens arepolypeptides, synthetic peptides and/or recombinant proteins associatedwith the presence of CSF to generate antibodies that recognize thepolypeptides associated with the presence of CSF. In one embodiment, theantibody is reactive with a native or “folded” protein. In anotherembodiment, an antibody is reactive with denatured protein (includingdetergent solubilized). Such antibodies include, but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments, Fabexpression libraries, or recombinant (e.g., chimeric, humanized, etc.)antibodies, as long as it can recognize the protein. Anti-bodies can beproduced by using a protein or peptide as the antigen according to aconventional antibody or antiserum preparation process.

Various procedures are used for the production of polyclonal antibodiesdirected against polypeptides associated with the presence of CSF. Forthe production of an antibody, various host animals are immunized byinjection with the polypeptides, synthetic peptides and/or recombinantproteins associated with the presence of CSF or a fragment thereofincluding but not limited to rabbits, mice, rats, sheep, goats, chicken,donkey, etc. In a specific embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels (e.g., aluminum hydroxide), surface activesubstances (e.g., lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Corynebacterium parvum).

For preparation of monoclonal antibodies directed toward polypeptides,synthetic peptides and recombinant proteins associated with the presenceof CSF, it is contemplated that a technique that provides for theproduction of antibody molecules by continuous cell lines in culturewill find use herein. These include, but are not limited to, thehybridoma technique originally developed by Kohler and Milstein, as wellas the trioma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique to produce human monoclonal antibodies.

In additional embodiments, monoclonal antibodies are produced ingermfree animals. Furthermore, it is contemplated that human antibodieswill be generated by human hybridomas or by transforming human B cellswith EBV virus in vitro.

In addition, it is contemplated that techniques described for theproduction of single chain antibodies will find use in producing singlechain antibodies. An additional embodiment utilizes the techniquesdescribed for the construction of Fab expression libraries to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity.

In other embodiments, contemplated are recombinant antibodies orfragments thereof to polypeptides associated with the presence of CSF.Recombinant antibodies include, but are not limited to, humanized andchimeric antibodies. Methods for generating recombinant antibodies areknown in the art.

It is contemplated that a technique suitable for producing antibodyfragments will find use in generating antibody fragments that containthe idiotype (antigen binding region) of the antibody molecule. Forexample, such fragments include but are not limited to: F(ab′)2 fragmentthat can be produced by pepsin digestion of the antibody molecule; Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and Fab fragments that can be generated by treatingthe antibody molecule with papain and a reducing agent.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. The immunogenic peptide may be provided free of the carriermolecule used in any immunization protocol. For example, if the peptidewas conjugated to KLH, it may be conjugated to BSA, or used directly, ina screening assay.

The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of polypeptides associatedwith the presence of CSF (e.g., for Western blotting), measuring levelsthereof in appropriate biological samples, etc. The antibodies can beused to detect polypeptides associated with the presence of CSF in abiological sample from an individual. The biological sample is abiological fluid, such as, but not limited to, tissue, blood, serum,plasma, urine, nasal and ear effluents, saliva, sweat, tears and thelike. In one embodiment, the sample is from an individual suspected ofhaving a brain injury, such as mild traumatic head injury receivedduring participation in sporting events, auto accidents, militaryactivity and motorcycle accidents. The test would be most useful whenthe injury is mild to moderate in severity. More severe head injuryincluding penetrating injuries generally already receive the necessarylevel of medical attention. Diagnosis of traumatic brain injuriesgenerally requires a short neurological exam (the GCS). The precisedesignations of mild and moderate are sometimes hard to objectivelyidentify without a recent baseline, pre injury test. Other injuries ortreatments (sedative, anesthetics, etc) can interfere with the test. Thecurrent set of antigens can represent “biomarkers” which could be usedto “fingerprint” the existence and severity of a head injury. A rapidtest that is qualitative or quantitative of the existence of a subset ofthese antigens in blood or other bodily fluids (sweat, urine, saliva,etc.) can be used as a measure of the severity of an injury incombination with a GCS or any such neurological exam. Often the severityof a mild to moderate head injury is not know and to what degree theperson should continue to engage in critical activities (i.e. continuingto participate in a sporting event, continue to work or drive a vehicle,remain in the combat arena, continue to assume a command position incombat, operate heavy machinery, etc.). A more objective test of bloodborne or secreted proteins normally found enriched only in the CSF wouldrepresent a diagnostic test of injury.

The biological samples can then be tested directly for the presence ofpolypeptides associated with the presence of CSF using an appropriatestrategy (e.g., ELISA or radioimmunoassay) and format (e.g., microwells,dipstick (e.g., as described in International Patent Publication WO93/03367), etc. Alternatively, proteins in the sample can be sizeseparated (e.g., by polyacrylamide gel electrophoresis (PAGE), in thepresence or not of sodium dodecyl sulfate (SDS) Triton, Noniodet (orother ionic or non-ionic detergents), and the presence of a CSF antigendetected by immunoblotting (Western blotting). Immunoblotting techniquesare generally more effective with antibodies generated against a peptidecorresponding to an epitope of a protein, and hence, are particularlysuited to the present disclosure.

The correlation step mentioned above may be implemented qualitatively orquantitatively, for example in a fluorophoric or colorimetric assay.

Kits and Devices:

Also provided are kits and devices for determining whether a samplecontains polypeptides associated with the presence of CSF. Thediagnostic kits and devices are produced in a variety of ways. In someembodiments, the kits and devices contain at least one reagent forspecifically detecting a polypeptide associated with the presence ofCSF. In specific embodiments, the kits and devices contain multiplereagents for detecting polypeptides associated with the presence of CSF.In other embodiments, the reagents are antibodies that preferentiallybind polypeptides associated with the presence of CSF. The test canproduce a single result indicating the presence of CSF from a number(2-10) of tests for multiple antigens or each test can produce adifferent evident result that can be interpreted to indicate thepresence or absence of CSF.

In some embodiments, the kit or device contains instructions fordetermining whether the sample contains polypeptides associated with thepresence of CSF. In specific embodiments, the instructions specify thatpresence or absence of CSF is determined by detecting the presence orabsence of polypeptides associated with the presence of CSF in a samplefrom the subject.

In some embodiments, the kits and devices include ancillary reagentssuch as buffering agents, protein stabilizing reagents, and signalproducing systems (e.g., fluorescence generating systems such as FRETsystems). The test kit or device is packaged in a suitable manner,typically with the elements in a single container or various containersas necessary, along with a sheet of instructions for carrying out thetest. In some embodiments, the kits or devices also include a positivecontrol sample. In further embodiments, the kit or device containscomparative reference material to interpret the presence or absence ofpolypeptides associated with the presence of CSF according to intensity,color spectrum, or other physical attribute of an indicator.

The need for a rapid, reproducible, sensitive and simple diagnostictest, which can be used in the health care for diagnosing CSF, is ofmajor importance. Such a test has the obvious advantage over theexisting laboratory tests, i.e., immunofixation electrophoresis,enzyme-linked immunosorbant assay (ELISA) and immunoblotting, in that itcan be performed immediately beside the patient giving a result in a fewminutes of time instead of several days when the sample is sent foranalysis to a laboratory. A lateral flow immunochromatographic test maybe utilized for making a diagnostic kit for the detection of CSF inbiological fluids.

In one embodiment, a device includes a solid phase comprising a firstregion comprising a mobile indicator suitable for binding a CSF antigen,and a second region comprising a fixed indicator suitable for bindingthe CSF antigen.

In one embodiment, a lateral flow device comprises a test stripoptionally with a plastic test cassette. Antibodies are attached tothree different zones on the membrane; a sample zone (S) containing afirst monoclonal antibody to a polypeptide associated with the presenceof CSF; a test zone (T) that contains a second monoclonal or polyclonalantibody to polypeptides associated with the presence of CSF immobilizedto the membrane; and a control zone (C), which contains a controlantibody, for example, an immobilized rabbit anti-mouse antibody. Thefirst monoclonal antibody in the sample (S) zone may be conjugated to amobile particle, for example, a colored latex particle or a goldparticle. Alternatively, the first monoclonal antibody is conjugated toa chromophoric indicator, such as a fluorescent molecule or tag (GreenFluorescent Protein (GFP) or FP orthologs mutants and other naturallyoccurring GFP-like fluorescent and chromo proteins, fluorescein (andorthologs), rhodamine (and orthologs), Cy3, Cy5, Cy2, Cy7, Cy8, Alexa®dyes, Texas Red, and the like).

An exemplary device is implemented utilizing an immunochromatographictest based on the use of two monoclonal antibodies. Sample is added tothe S-zone, and if the polypeptide associated with the presence of CSFis present, it binds to the first monoclonal antibody to form apolypeptide-conjugate-complex. This complex migrates chromatographicallyon the membrane, and when it reaches the immobilized antibody in theT-zone, agglutination takes place and a blue colored band is formed.

Briefly and in one embodiment, the first monoclonal antibody isconjugated to a mobile particle, for example, gold or latex beads. Thesebeads have the intrinsic color of either being red (for gold) or cancome in different colors if using latex beads. When the sample isapplied on the “S-zone”, the marker, a polypeptides associated with thepresence of CSF if present in the sample, binds to the first monoclonalantibody that is conjugated to the beads and then because of the lateralflow absorbent pad on which the beads are placed, the complex(beads+antibody+polypeptide if present in the sample) migrateslaterally. Once the complex reaches the “T-zone” where the secondantibody is immobilized on the strip, the marker that is now migratingwith the complex binds to the second immobilized antibody. As the secondantibody is stationary/fixed/immobilized, the whole complex gets trappedand as the complex now contains colored beads, the immobilized T-zoneline lights up according to the beads that are used (red for gold ordifferent colors {like blue} if latex beads are used). The excesscomplex sample migrates to the end of the strip and at the “C-zone” thefirst antibody conjugated to the beads is trapped byimmobilized/fixed/stationary rabbit-anti mouse antibody and gives acolored line indicating that the test is complete). Thus, a colored bandindicates a positive result. No band in the T-zone is significant for anegative result. The immobilized polyclonal antibody in the C-zone willbind the latex conjugate with both positive and negative samples. Thisblue band assures a correct test performance.

In practice, the kits and devices are utilized in a variety of clinicalsettings to determine the presence of CSF in a sample.

The invention is illustrated by the following non-limiting examples.

Examples

CSF-specific antigens newly identified herein include Isoform 1 ofNeural cell adhesion molecule-like (Accession Number gi|62088238)protein; Chain A, Human Mesotrypsin Complexed With Bovine PancreaticTrypsin Inhibitor (Bpti) (Accession number gi|162330095); CNTN2Contactin-2 (Accession Number gi|4827022); CNTN1 Isoform 2 ofContactin-1 (Accession Number gi:28373119); cDNA highly similar toSPARC-like protein 1 (Accession Number: gi|194388050); NRCAM protein(Neuronal cell adhesion molecule)[Homo sapiens] possibly slightly longerfragment (˜96 kDa) (Accession Number: gi|68534652 and gi|109731501);NCAM2 Neural cell adhesion molecule 2 (Accession Number gi|119630409);SERPINA3 serpin peptidase inhibitor, clade A, member 3 precursor/Isoform1 of Alpha-1-antichymotrypsin/growth-inhibiting protein 25 [Homosapiens] or slightly longer fragment of alpha-1-antichymotrypsinprecursor (Accession Number gi|46981961); AGT Angiotensinogen (AccessionNumber gi|553181); Angiotensinogen precursor (Serpin A8) (AccessionNumber gi|4557287); unnamed protein product also called immunoglobulinsuperfamily, member 4B; in humans, also called cell adhesion molecule 3;possible fragment (Accession Number gi|187608363); cDNA FLJ59893,dickkopf homolog 3 precursor (Accession Number gi|40548389); SERPINF1serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin,pigment epithelium derived factor), member 1 isoform 4 [Pan troglodytes]factor (Accession Number gi|15988024); GC Vitamin D-binding proteinPREDICTED: vitamin D-binding protein [Pan troglodytes] (Accession Number181482); CD14 Human monocyte antigen CD14 (CD14) (Accession Numbergi|117646212); CADM3 Homo sapiens cell adhesion molecule 3 (CADM3),transcript variant 1 (Accession Number gi|90080503; gi|187608363(human); Neural cell adhesion molecule variant (Accession Numbergi62088238); CLU cDNA FLJ57622, highly similar to Clusterin (Accessionnumber gi|189054091); protein highly similar to Clusterin (Accessionnumber gi|193787502); LMAN2 Vesicular integral-membrane protein VIP36(Accession number gi|157834800); superoxide dismutase 3, extracellularprecursor (Accession number gi|118582275); fibrin alpha C term fragment(Accession number gi|223057); KLK6 Isoform 1 of Kallikrein-6 (Accessionnumber gi|21465970); APCS Serum amyloid P-component/Chain A, TheStructure Of Pentameric Human Serum Amyloid P Component (Accessionnumber gi|576259); FAM3C Protein FAM3C/family with sequence similarity3, member C precursor [Homo sapiens] note=“predicted osteoblast protein;interleukin-like EMT inducer (Accession number gi|55629272); Chain A,Human Kallikrein 6 (Hk6) Active Form With Benzamidine Inhibitor(Accession number gi|21465970); unnamed protein product [Macacafascicularis] also called immunoglobulin superfamily, member 4B; inhumans, also called cell adhesion molecule 3; possible fragment(Accession number gill 87608363); a CSF-enriched phosphorylated ordephosphorylated form of the foregoing CSF antigens; or a combination oftwo or more of the foregoing CSF antigens.

The terms “a” and “an” herein do not denote a limitation of quantity,but rather denote the presence of at least one of the referenced item.

All ranges disclosed herein are inclusive and combinable. While theinvention has been described with reference to a preferred embodiment,it will be understood by those skilled in the art that various changesmay be made and equivalents may be substituted for elements thereofwithout departing from the scope of the invention. In addition, manymodifications may be made to adapt a particular situation or material tothe teachings of the invention without departing from essential scopethereof. Therefore, it is intended that the invention not be limited tothe particular embodiment disclosed as the best mode contemplated forcarrying out this invention, but that the invention will include allembodiments falling within the scope of the appended claims.

All cited patents, patent applications, and other references areincorporated herein by reference in their entirety.

1. A device for detection of the presence or absence of cerebrospinalfluid in a sample, comprising a sample application region, a samplelabeling region comprising a first antibody to a CSF-enriched protein,wherein the first antibody is conjugated to a mobile particle; a sampledetection region comprising a second antibody to the CSF-enrichedprotein, wherein the second antibody is fixed to the sample detectionregion, wherein the presence of a detectable band in the sampledetection region indicates the presence of cerebrospinal fluid in thesample, wherein the CSF-enriched protein is selected from the groupconsisting of CNTN2 Contactin-2 precursor (SEQ ID NO: 3; AccessionNumber gi|4827022); cDNA FLJ59893, dickkopf homolog 3 precursor (SEQ IDNO: 13; Accession Number gi|40548389); CADM3 Homo sapiens cell adhesionmolecule 3 (CADM3), transcript variant 1 (SEQ ID NO: 17; Neural celladhesion molecule variant (SEQ ID NO: 19; Accession Number gi:62088238);a CSF-enriched phosphorylated or dephosphorylated form of the foregoingCSF antigens; and a combination of two or more of the foregoing CSFantigens.
 2. The device of claim 1, wherein the device comprises two tofour different antibodies that each specifically binds a differentCSF-enriched protein.
 3. The device of claim 2, wherein the deviceprovides a single combined result, or the device provides an individualresult for each antibody.
 4. The device of claim 2, wherein eachantibody is employed at a subthreshold level.
 5. The device of claim 1,further comprising quantitating the level of CSF-enriched protein in thesample.
 6. The device of claim 1, wherein the first antibody to aCSF-enriched protein binds a post-translational modification in theCSF-enriched protein.
 7. A method for detecting the presence or absenceof CSF in a sample, comprising contacting the sample with a bindingpartner specific for a CSF-enriched protein, and detecting bindingpartner-CSF-enriched protein complexes if present, wherein the presenceof detectable complexes indicates the presence of CSF in the sample;wherein the CSF-enriched protein is selected from the group consistingof CNTN2 Contactin-2 precursor (SEQ ID NO: 3; Accession Numbergi|1827022); NRCAM protein (Neuronal cell adhesion molecule)[Homosapiens] possibly slightly longer fragment (˜96 kDa) SEQ ID NO: 7;gi|109731501); cDNA FLJ59893, dickkopf homolog 3 precursor (SEQ ID NO:13; Accession Number gi|40548389); CADM3 Homo sapiens cell adhesionmolecule 3 (CADM3), transcript variant 1 (SEQ ID NO: 17; Neural celladhesion molecule variant (SEQ ID NO: 19; Accession Number gi:62088238);a CSF-enriched phosphorylated or dephosphorylated form of the foregoingCSF antigens; and a combination of two or more of the foregoing CSFantigens.
 8. The method of claim 7, wherein the binding partnercomprises a detectable label.
 9. The method of claim 8, furthercomprising quantitating the amount of binding partner-CSF-enrichedprotein complexes in the sample.
 10. The method of claim 7, wherein thebinding partner is an antibody and detecting comprises differentialantibody binding.
 11. The method of claim 7, wherein the binding partneris an antibody and detecting comprises an in situ immunoassay.
 12. Themethod of claim 7, wherein the sample is tissue, blood, serum, plasma,urine, nasal and ear effluents, saliva, sweat, or tears.
 13. The methodof claim 7, wherein the sample is from an individual suspected of havinga brain injury.
 14. The method of claim 7, comprising contacting thesample with two to ten different antibodies that each specifically bindsa different CSF-enriched protein.
 15. The method of claim 14, whereineach antibody is employed at a subthreshold level.
 16. The method ofclaim 7, wherein the binding partner is an antibody and the assaycomprises comprising contacting the sample with four to ten differentantibodies that each specifically binds a different CSF-enrichedprotein, and wherein a positive test does not require binding to allantibodies.
 17. The method of claim 7, wherein the binding partner is anantibody that binds a post-translational modification in theCSF-enriched protein.
 18. A method for the detection of a reactant in abody fluid, tissue or microorganism, comprising contacting the bodyfluid, tissue or microorganism with two or more antibodies, wherein eachantibody specifically reacts with an antigen in the reactant, whereinreaction with each individual antibody does not indicate a positive testfor the reactant, and wherein reaction with the two or more antibodiesindicates a positive test for the reactant.
 19. The method of claim 18,wherein the two or more antibodies bind CSF-enriched proteins and thetest is suitable to indicate the presence of CSF in a sample.